Screening of 10 DFNB Loci Causing Autosomal Recessive Non-Syndromic Hearing Loss in Two Iranian Populations Negative for GJB2 Mutations.

Background
Autosomal recessive non-syndromic hearing loss (ARNSHL), one of the global public health concerns, is marked by a high degree of genetic heterogeneity. The role of GJB2, as the most common cause of ARNSHL, is only <20% in the Iranian population. Here, we aimed to determine the relative contribution of several apparently most common loci in a cohort of ARNSHL Iranian families that were negative for the GJB2 mutations.


Methods
Totally, 80 Iranian ARNSHL families with 3 or more affected individuals from Isfahan and Hamedan provinces, Iran were enrolled in 2017. After excluding mutations in the GJB2 gene via Sanger sequencing, 60 negative samples (30 families from each province) were analyzed using homozygosity mapping for 10 ARNSHL loci.


Results
Fourteen families were found to be linked to five different known loci, including DFNB4 (5 families), DFNB2 (3 families), DFNB7/11 (1 family), DFNB9 (2 families) and DFNB3 (3 families).


Conclusion
Despite the high heterogeneity of ARNSHL, the genetic causes were determined in 23.5% of the studied families using homozygosity mapping. This data gives an overview of the ARNSHL etiology in the center and west of Iran, used to establish a diagnostic gene panel including most common loci for hearing loss diagnostics.


Introduction
Hearing loss (HL) is the most frequent form of sensory impairment in humans, with approximately 1-2 in 1000 infants being born with a seri-ous HL (https://www.gov.uk/guidance/newbornhearing-screening-programme-overview). Over 70% of pre-lingual HL cases can be classified as non-syndromic HL (NSHL), where the hearing deficit is the only sign (1). HL can be inherited as autosomal recessive, autosomal dominant, mitochondrial, X-and Y-linked. Autosomal recessive mode of inheritance (ARNSHL) makes up 80% of the NSHL cases. ARNSHL is highly heterogeneous, for which over 100 mapped loci are known. The autosomal recessive loci are called DFNB followed by a number corresponding to the order that the locus was first explained; DFNB1 to DFNB105 have been reported so far (http://hereditaryhearingloss.org).
However, more than seventy different DFNB loci have been mapped for ARNSHL by homozygosity mapping, an effective method to locate genes causing ARNSHL in large consanguineous families (2). The genetic etiology of ARNSHL in Iran has been studied by a number of independent studies with a special focus on the certain locus DFNB1 (GJB2), as the most common cause of HL all over the world (3). Variants in the GJB2 (NM_004004.5) could explain the etiology of ARNSHL in 4% to 35% of patients from different origin, suggesting that GJB2 gene mutations account for a part of ARNSHL in Iran. (4,5). On the other hand, Iran is a large country with many ethnic groups and high rate of the consanguineous marriages (38.6% on average) (6). Thus, mutations in certain loci may be increased within some ethnic groups (7). Therefore, populationspecific studies are necessary to identify other common loci and to determine the etiology of ARNSHL based on ethnicity. Until now, little data is available on the contribution of some frequent loci among the Iranian ARNSHL; and have mostly focused on mixed populations from different provinces (8). Mutations in at least 40 genes have been explained to cause ARNSHL in Iran. However, the contribution of their mutations does not appear to occur at the same frequencies across different ethnic groups (9). We applied the homozygosity mapping strategy to identify the relative contribution of 10 DFNB loci to ARNSHL in the studied cohort including 60 ARNSHL families, which were negative for GJB2 mutations, from Isfahan and Hamedan provinces for the first time. This data could be applied to design a cheap and accurate panel for common genes causing ARNSHL in certain regions of Iran and would lead to affordable testing and improved management of HL (10).

Families and clinical evaluations
Totally, 60 unrelated consanguineous families with 3 or more hearing impaired patients and negative for GJB2 mutations were recruited from Isfahan and Hamedan provinces in the center and west of Iran in 2017. The Ethics Committee of the Isfahan University of Medical Sciences approved this project. All family members signed informed written consent prior to recruitment. They met the following criteria: 1) confirmation of HL by Pure tone audiometry (PTA) from 250-8000 Hz 2) the autosomal recessive inheritance deduced through pedigree analysis 3) existence of three or more affected members within the pedigree. A complete clinical investigation was performed to exclude environmental exposures and to determine the presence of syndromic findings in each family.

DNA extraction
Genomic DNA was extracted using Prime Prep Genomic DNA Extraction kit from blood (GeNet Bio, Korea) according to the manufacturer's instruction. Qualitative and quantitative assessment of genomic DNA was checked using 1.2% agarose gel and Nanospec cube biophotometer (Nanolytik®, Dusseldorf, Germany).

SLINK analysis and selection of DFNB loci
Power of the pedigrees for linkage analysis was simulated by calculating SLINK, using FastSLink (ver.2.51) option of Easy linkage plus version 5.05 software to predict the potential LOD score in a given family (11). Based on the literature review of the most frequent loci, both globally and regionally, 10 loci were selected for screening. Screening sort tandem repeat (STR) markers were selected based on their physical distance found at NCBI UniSTS and NCBI Map Viewer

Families and clinical data
After excluding mutations in the GJB2 gene, 60 Iranian families segregating (ARNSHL), from Isfahan and Hamedan provinces (30 families from each province) in the center and west of Iran, were screened for 10 ARNSHL loci. Totally, 634 individuals were studied 245 of whom were patients, with ages ranging from 6 months to 52 yr. For 45 families, PTA was consistent with profound HL (≥80 dB), 12 families showed severe HL (61-80 dB), whereas the 3 remaining families showed moderate HL (41-60 dB).

SLINK calculation, genotyping and linkage analysis
Totally The family members were individually genotyped for these markers. After genotyping of STR markers and linkage analysis, 14 out of the 60 families, negative for GJB2 mutations, showed linkage to five different loci ( Table 2). DFNB4 was the most frequent locus in the studied ARNSHL series in both provinces (36.9% of the etiology). Three out of 30 (10%) and 2 of 30 (6.6%) families were linked to DFNB4 in Isfahan and Hamedan provinces, respectively. DFNB2, DFNB3, and DFNB9 were ranked next after DFNB4. Table 3 shows the linked families and the maximum values for SLINK, two-point and multipoint LOD scores. One family (3.3%) from every single province was linked to DFNB9. One family from Hamedan Province was linked to DFNB7/11 (7.7% of the etiology). The haplotypes of 3 selected linked families are shown in Fig. 1(a-c). We did not find linkage to the other loci (DFNB21, DFNB24, DFNB59, DFNB63 and FNB53) among the studied families.

Discussion
We determined the contribution of 10 DFNB loci to HL in 60 Iranian families affected with ARNSHL and negative for the GJB2 mutations. We have obtained 20% and 26% involvement of GJB2 mutations in ARNSHL in Hamedan and Isfahan provinces, respectively (unpublished data).    (15). In a recent study 12 out of 121 (9.9%) families were linked to DFNB4 (16). Thus, our result shows that DFNB4 contributes mainly to ARNSHL in the west and center of Iran and is ranked second after DFNB1, which is in agreement with previous studies reported from Iran ( Table 4). The next most frequently linked locus in our cohort was DFNB2, which accounts for about 6.6% of HL etiology in Hamedan and 3.3% in Isfahan. The related gene MYO7A, encoding myosin VIIA, is an action-based molecular motor converting energy from ATP hydrolysis into mechanical force. It also interacts with actin to participate in the formation of the mechanotransduction complex, which is critical for detecting sound (17).  (19). Similarly, Sloan-Heggen et al. (20) using a custom targeted genomic enrichment method in a cohort of 302 GJB2-negative Iranian families, found the DFNB2 contribution to be 5%, which is in accordance with our results ( Table 4).
Our data show that one out of 30 families (3.3%) from Isfahan province and two out of 30 families (6.6%) from Hamedan were linked to DFNB3. The DFNB3 locus was first identified in the village of Indonesia, Bengkala, with a frequency of 9.4% among the inbred population (21). Since then, many mutations have been reported from different countries such as India, Iran, Turkey, and Brazil (22,23). In Pakistan, DFNB3 is the third locus for ARNSHL accounting for 5% of ARNSHL (24). In a previous study on 40 Iranian ARNSHL families from Qom and Markazi provinces, from center of Iran, 2 families were linked to DFNB3 (5.8%) (25). Therefore, the locus could be one common cause of ARNSHL both in the west and center of Iran.
In the present study, one family (3.3%) from every single province was linked to DFNB9 (OTOF). Mutations in the OTOF gene (named as homolo-gy to Ferlin (Fer-1)) encoding otoferlin at DFNB9 results in ARNSHL which is sometimes associated with auditory neuropathy. Otoferlin is a member of the ferlin protein family; its role is in vesicle recycling and efficient and linear encoding of low-intensity stimulate the synapse between inner hair cells and auditory nerve fibers (26). OTOF mutations have been reported from many countries such as Pakistan, Spain, Italy, and Japan with different mutation spectrum (26,27). The frequency of the OTOF gene mutations has been reported to be 2.7% in Iran (28). It was screened 37 Iranian ARNSHL families from 7 different provinces for 15 loci using linkage analysis and found 1 family (2.7%) to be linked to the DFNB9 locus. Similarly, in our present study, one family (3.3%) from each of the two provinces was linked to DFNB9 (OTOF). Our study involves 3 families with moderate to severe HL highlighted by a "U" shaped audiogram. DFNB21 and DFNB93 are related to this audio profile (18,29). Interestingly, none of the 3 families was linked to DFNB21. Thus, the next step for these families involves investigating the DFNB93 contribution (30).  (7,33). The identified mutations and their characteristics are listed in Table 5. The large size of some of the related genes hinders detection of their related mutations (34). However, the remaining families have been considered for sequencing of the related genes.
In the present study, despite the high heterogeneity of ARNSHL, we could detect the genetic etiology in 6 out of 30 (20%) and 8 out of 30 (26.6%) ARNSHL families, negative for GJB2 mutations, from Isfahan and Hamedan provinces, respectively. Thus, nearly 23.5% of ARNSHL families negative for GJB2 mutations are linked to 5 loci including DFNB4, DFNB2, DFNB3, DFNB9 and DFNB7/11. The remaining genes and loci could be either rare or yet to be identified (2,28). The study is in progress by subjecting some of the remaining families to the nextgeneration sequencing, which is a rapid and costeffect method.

Conclusion
Using homozygosity mapping, we detected 23.5% of the genetic etiology of GJB2-mutation negative ARNSHL in Isfahan and Hamedan provinces. This finding is interesting in view of the extreme genetic heterogeneity of ARNSHL (with over 50 genes discovered so far). Investigation of a limited number of genes could lead to an acceptable level of diagnostic yield.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.